Abstract
This chapter describes the assay method, purification, and properties of diacetyl reductase. Diacetyl reductase is assayed by measuring the decrease in absorbance at 340 nm caused by nicotinamide adenine dinucleotide dehydrogenase (NADH) oxidation during diacetyl reduction to acetoin. The assay is performed at 25°C in 0.1 M Na 2 –K phosphate buffer. The test solution contains the enzyme preparation, NADH, and diacetyl in a total volume of 3 ml. Crude extracts have NADH-oxidase activity and assays are performed using a control without diacetyl. Beef liver diacetyl reductase has a K diacety1 m lower than that from other sources and shows activation by an excess of substrate; the concentrations of 4 m M diacetyl in the reaction mixture are recommended for the enzyme. Three different enzymes described as diacetyl reductases are isolated: enzymes from the beef liver, the pigeon liver, and Escherichia coli . The steps involved in the purification of the enzyme are (1) extraction, (2) acetone precipitation, (3) diethylaminoethyl (DEAE)-cellulose chromatography, and (4) electrofocusing. Diacetyl reductase activity is distributed in the beef liver between the soluble and mitochondrial fractions.
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